Abstract:

The invention provides compositions and methods for selectively reducing
the expression of a gene product from a desired target gene, as well as
treating diseases caused by expression of the gene. The method involves
introducing into the environment of a cell an amount of a double-stranded
RNA (dsRNA) such that a sufficient portion of the dsRNA can enter the
cytoplasm of the cell to cause a reduction in the expression of the
target gene. The dsRNA has a first oligonucleotide sequence that is
between 26 and about 30 nucleotides in length and a second
oligonucleotide sequence that anneals to the first sequence under
biological conditions. In addition, a region of one of the sequences of
the dsRNA having a sequence length of from about 19 to about 23
nucleotides is complementary to a nucleotide sequence of the RNA produced
from the target gene.

Claims:

1. A mammalian cell containing an isolated double stranded nucleic acid
comprising first and second oligonucleotide strands, each strand
comprising ribonucleotides and having a 5' terminus and a 3' terminus,
wherein said double stranded nucleic acid comprises blunt ends and each
of said first and said second strands consists of the same number of
nucleotide residues and is 25-30 nucleotides, wherein the ultimate and
penultimate residues of said 3' terminus of said first strand and the
ultimate and penultimate residues of said 5' terminus of said second
strand form one or two mismatched base pairs, and wherein said second
oligonucleotide strand comprises a sequence complementary to a target RNA
of a target gene and said isolated double stranded nucleic acid reduces
target gene expression in said mammalian cell.

2. The mammalian cell of claim 1, wherein said second oligonucleotide
strand of said isolated double stranded nucleic acid is complementary to
said target RNA along at least 19 nucleotides of said second
oligonucleotide strand length.

3. The mammalian cell of claim 1, wherein said double stranded nucleic
acid is cleaved endogenously in a mammalian cell to produce a double
stranded nucleic acid of a length in the range of 19-23 nucleotides in
length that reduces target gene expression.

4. The mammalian cell of claim 1, wherein each of said first and said
second strands of said isolated double stranded nucleic acid has a length
which is at least 26 and at most 30 nucleotides.

5. The mammalian cell of claim 1, wherein said first and second strands of
said isolated double stranded nucleic acid are, independently, 27
nucleotide residues in length.

6. The mammalian cell of claim 1, wherein the ultimate and penultimate
residues of said 3' terminus of said first strand and the ultimate and
penultimate residues of said 5' terminus of said second strand of said
isolated double stranded nucleic acid form two mismatched base pairs.

7. The mammalian cell of claim 1, wherein said nucleotides of each of said
first and second oligonucleotide strands are ribonucleotides.

8. The mammalian cell of claim 1, wherein said second strand is fully
complementary to the target RNA.

9. The mammalian cell of claim 1, wherein said isolated double stranded
nucleic acid comprises a modified nucleotide selected from the group
consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an
acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a
3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddI), a
2',3'-dideoxy-3'-thiacytidine (3TC), a
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a monophosphate nucleotide
of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine
(3TC) and a monophosphate nucleotide of
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a 4-thiouracil, a
5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2'-O-alkyl
ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-amino ribonucleotide,
a 2'-fluoro ribonucleotide, and a locked nucleic acid.

10. The mammalian cell of claim 1, wherein said isolated double stranded
nucleic acid comprises a phosphate backbone modification selected from
the group consisting of a phosphonate, a phosphorothioate, and a
phosphotriester.

11. The mammalian cell of claim 1, wherein the concentration of said
double stranded nucleic acid sufficient to reduce expression of the
target gene is selected from the group consisting of 1 nanomolar or less,
200 picomolar or less and 50 picomolar or less in the environment of said
cell.

12. The mammalian cell of claim 1, wherein said double stranded nucleic
acid reduces said target gene expression in a mammalian cell in vitro by
an amount (expressed by %) selected from the group consisting of at least
10%, at least 50% and at least 80%.

13. A mammalian cell containing an isolated double stranded nucleic acid
comprising first and second oligonucleotide strands, each strand
comprising ribonucleotides and having a 5' terminus and a 3' terminus,
wherein said double stranded nucleic acid comprises blunt ends and each
of said first and said second strands is 27 nucleotides in length,
wherein the ultimate and penultimate residues of said 3' terminus of said
first strand and the ultimate and penultimate residues of said 5'
terminus of said second strand form two mismatched base pairs, and
wherein said second oligonucleotide strand comprises a sequence
complementary to a target RNA of a target gene and said isolated double
stranded nucleic acid reduces target gene expression in said mammalian
cell.

14. The mammalian cell of claim 13, wherein said double stranded nucleic
acid is cleaved endogenously in a mammalian cell to produce a double
stranded nucleic acid of a length in the range of 19-23 nucleotides in
length that reduces target gene expression.

15. The mammalian cell of claim 13, wherein said isolated double stranded
nucleic acid comprises a modified nucleotide selected from the group
consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an
acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a
3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddI), a
2',3'-dideoxy-3'-thiacytidine (3TC), a
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a monophosphate nucleotide
of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine
(3TC) and a monophosphate nucleotide of
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a 4-thiouracil, a
5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2'-O-alkyl
ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-amino ribonucleotide,
a 2'-fluoro ribonucleotide, and a locked nucleic acid.

16. The mammalian cell of claim 13, wherein said isolated double stranded
nucleic acid comprises a phosphate backbone modification selected from
the group consisting of a phosphonate, a phosphorothioate, and a
phosphotriester.

17. A mammalian cell containing an isolated double stranded nucleic acid
comprising first and second oligonucleotide strands, each strand
comprising ribonucleotides and having a 5' terminus and a 3' terminus,
wherein said double stranded nucleic acid comprises blunt ends and each
of said first and said second strands consists of the same number of
nucleotide residues and is 25-30 nucleotides, wherein the ultimate and
penultimate residues of said 5' terminus of said first strand and the
ultimate and penultimate residues of said 3' terminus of said second
strand form one or two mismatched base pairs, and wherein said second
oligonucleotide strand comprises a sequence complementary to a target RNA
of a target gene and said isolated double stranded nucleic acid reduces
target gene expression in said mammalian cell.

18. The mammalian cell of claim 17, wherein the ultimate and penultimate
residues of said 5' terminus of said first strand and the ultimate and
penultimate residues of said 3' terminus of said second strand of said
isolated double stranded nucleic acid form two mismatched base pairs.

19. The mammalian cell of claim 17, wherein said double stranded nucleic
acid is cleaved endogenously in a mammalian cell to produce a double
stranded nucleic acid of a length in the range of 19-23 nucleotides in
length that reduces target gene expression.

20. The mammalian cell of claim 17, wherein said isolated double stranded
nucleic acid comprises a modified nucleotide selected from the group
consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an
acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a
3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddI), a
2',3'-dideoxy-3'-thiacytidine (3TC), a
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a monophosphate nucleotide
of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine
(3TC) and a monophosphate nucleotide of
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a 4-thiouracil, a
5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2'-O-alkyl
ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-amino ribonucleotide,
a 2'-fluoro ribonucleotide, and a locked nucleic acid.

21. The mammalian cell of claim 17, wherein said isolated double stranded
nucleic acid comprises a phosphate backbone modification selected from
the group consisting of a phosphonate, a phosphorothioate, and a
phosphotriester.

22. A mammalian cell containing an isolated double stranded nucleic acid
comprising first and second oligonucleotide strands, each strand
comprising ribonucleotides and having a 5' terminus and a 3' terminus,
wherein said double stranded nucleic acid comprises blunt ends and each
of said first and said second strands is 27 nucleotides in length,
wherein the ultimate and penultimate residues of said 5' terminus of said
first strand and the ultimate and penultimate residues of said 3'
terminus of said second strand form two mismatched base pairs, and
wherein said second oligonucleotide strand comprises a sequence
complementary to a target RNA of a target gene and said isolated double
stranded nucleic acid reduces target gene expression in said mammalian
cell.

23. The mammalian cell of claim 22, wherein said double stranded nucleic
acid is cleaved endogenously in a mammalian cell to produce a double
stranded nucleic acid of a length in the range of 19-23 nucleotides in
length that reduces target gene expression.

24. The mammalian cell of claim 22, wherein said isolated double stranded
nucleic acid comprises a modified nucleotide selected from the group
consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an
acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a
3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddI), a
2',3'-dideoxy-3'-thiacytidine (3TC), a
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a monophosphate nucleotide
of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine
(3TC) and a monophosphate nucleotide of
2',3'-didehydro-2',3'-dideoxythymidine (d4T), a 4-thiouracil, a
5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2'-O-alkyl
ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-amino ribonucleotide,
a 2'-fluoro ribonucleotide, and a locked nucleic acid.

25. The mammalian cell of claim 22, wherein said isolated double stranded
nucleic acid comprises a phosphate backbone modification selected from
the group consisting of a phosphonate, a phosphorothioate, and a
phosphotriester.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001]The present application is a division of U.S. patent application No.
12/137,914 filed 12 Jun. 2008, which in turn is a division of U.S. patent
application No. 11/079,476 filed 15 Mar. 2005, which in turn is related
to and claims priority under 35 U.S.C. §119(e) to U.S. provisional
patent application No. 60/553,487 filed 15 Mar. 2004. Each application is
incorporated herein by reference.

FIELD OF THE INVENTION

[0003]This invention pertains to compositions and methods for
gene-specific inhibition of gene expression by double-stranded
ribonucleic acid (dsRNA) effector molecules. The compositions and methods
are useful in modulating gene expression in a variety of applications,
including therapeutic, diagnostic, target validation, and genomic
discovery.

[0005]In Drosophila cells and cell extracts, dsRNAs of 150 bp length or
greater were seen to induce RNA interference while shorter dsRNAs were
ineffective (Tuschl et al., 1999, Genes & Dev., 13:3191-3197). Long
double-stranded RNA, however, is not the active effecter molecule; long
dsRNAs are degraded by an RNase III class enzyme called Dicer (Bernstein
et al., 2001, Nature, 409:363-366) into very short 21-23 bp duplexes that
have 2-base 3'-overhangs (Zamore et al., 2000, Cell, 101:25-33). These
short RNA duplexes, called siRNAs, direct the RNAi response in vivo and
transfection of short chemically synthesized siRNA duplexes of this
design permits use of RNAi methods to suppress gene expression in
mammalian cells without triggering unwanted interferon responses
(Elbashir et al., 2001, Nature, 411:494-498). The antisense strand of the
siRNA duplex serves as a sequence-specific guide that directs activity of
an endoribonuclease function in the RNA induced silencing complex (RISC)
to degrade target mRNA (Martinez et al., 2002, Cell, 110:563-574).

[0006]In studying the size limits for RNAi in Drosophila embryo extracts
in vitro, a lower threshold of around 38 bp double-stranded RNA was
established for activation of RNA interference using exogenously supplied
double-stranded RNA and duplexes of 36, 30, and 29 bp length were without
effect (Elbashir et al., 2001, Genes & Dev., 15:188-200). The short
30-base RNAs were not cleaved into active 21-23-base siRNAs and therefore
were deemed inactive for use in RNAi (Elbashir et al., 2001, Genes &
Dev., 15:188-200). Continuing to work in the Drosophila embryo extract
system, the same group later carefully mapped the structural features
needed for short chemically synthesized RNA duplexes to function as
siRNAs in RNAi pathways. RNA duplexes of 21-bp length with 2-base
3'-overhangs were most effective, duplexes of 20, 22, and 23-bp length
had slightly decreased potency but did result in RNAi mediated mRNA
degradation, and 24 and 25-bp duplexes were inactive (Elbashir et al.,
2001, EMBO J., 20:6877-6888).

[0007]Some of the conclusions of these earlier studies may be specific to
the Drosophila system employed. Other investigators established that
longer siRNAs can work in human cells. However, duplexes in the 21-23-bp
range have been shown to be more active and have become the accepted
design (Caplen et al., 2001, Proc. Natl. Acad. Sci. USA, 98:9742-9747).
Essentially, chemically synthesized duplex RNAs that mimicked the natural
products that result from Dicer degradation of long duplex RNAs were
identified to be the preferred compound for use in RNAi. Approaching this
problem from the opposite direction, investigators studying size limits
for RNAi in C. elegans found that although a microinjected 26-bp RNA
duplex could function to suppress gene expression, it required a 250-fold
increase in concentration compared with an 81-bp duplex (Parrish et al.,
2000, Mol. Cell, 6:1077-1087).

[0008]Despite the attention given to RNAi research recently, the field is
still in the early stages of development. Not all siRNA molecules are
capable of targeting the destruction of their complementary RNAs in a
cell. As a result, complex sets of rules have been developed for
designing RNAi molecules that will be effective. Those having skill in
the art expect to test multiple siRNA molecules to find functional
compositions. (Ji et al. 2003) Some artisans pool several siRNA
preparations together to increase the chance of obtaining silencing in a
single study. (Ji et al. 2003) Such pools typically contain 20 nM of a
mixture of siRNA oligonucleotide duplexes or more (Ji et al. 2003),
despite the fact that a siRNA molecule can work at concentrations of 1 nM
or less (Holen et al. 2002). This technique can lead to artifacts caused
by interactions of the siRNA sequences with other cellular RNAs ("off
target effects"). (Scherer et al. 2003) Off target effects can occur when
the RNAi oligonucleotides have homology to unintended targets or when the
RISC complex incorporates the unintended strand from and RNAi duplex.
(Scherer et al. 2003) Generally, these effects tend to be more pronounced
when higher concentrations of RNAi duplexes are used. (Scherer et al.
2003)

[0009]In addition, the duration of the effect of an effective RNAi
treatment is limited to about 4 days (Holen et al. 2002). Thus,
researchers must carry out siRNA experiments within 2-3 days of
transfection with an siRNA duplex or work with plasmid or viral
expression vectors to obtain longer term silencing.

[0010]Additional physical studies are needed to more completely
characterize the structural requirements of RNAi active oligonucleotide
duplexes to identify more potent and longer lasting compositions and/or
methods that simplify site-selection difficulties. These studies should
also include a detailed analysis of the interferon response. Ideally,
such studies will be useful in identifying new RNAi active compounds that
are more potent, that simplify the site selection process, and decrease
"off target effects."

[0011]The invention provides RNAi compositions with increased potency,
duration of action, and decreased "off target effects" that do not
activate the interferon response and provides methods for their use. In
addition, the compositions ease site selection criteria and provide a
duration of action that is about twice as long as prior known
compositions. These and other advantages of the invention, as well as
additional inventive features, will be apparent from the description of
the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0012]The invention provides improved compositions and methods for
selectively reducing the expression of a gene product from a desired
target gene in a eukaryotic cell, as well as for treating diseases caused
by the expression of the gene. The method involves introducing into the
environment of a cell an amount of a double-stranded RNA (dsRNA) such
that a sufficient portion of the dsRNA can enter the cytoplasm of the
cell to cause a reduction in the expression of the target gene. The dsRNA
has a first oligonucleotide sequence that is between 26 and about 30
nucleotides in length and a second oligonucleotide sequence that anneals
to the first sequence under biological conditions, such as the conditions
found in the cytoplasm of a cell. In addition, a region of one of the
sequences of the dsRNA having a sequence length of from about 19 to about
23 nucleotides is complementary to a nucleotide sequence of the RNA
produced from the target gene. A dsRNA composition of the invention is at
least as active as any isolated 19, 20, 21, 22, or 23 basepair sequence
that is contained within it. Pharmaceutical compositions containing the
disclosed dsRNA compositions are also contemplated. The compositions and
methods give a surprising increase in the potency and duration of action
of the RNAi effect. Although the invention is not intended to be limited
by the underlying theory on which it is believed to operate, it is
thought that this increase in potency and duration of action are caused
by the fact the dsRNA serves as a substrate for Dicer which appears to
facilitate incorporation of one sequence from the dsRNA into the RISC
complex that is directly responsible for destruction of the RNA from the
target gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 provides a comparison of RNAi efficacy using several dsRNAs
having variable length and formats including a two nucleotide 3' overhang
(+2), a two nucleotide 5' overhang (-2), and blunt ends (+0). The
sequences are disclosed in the Example 2. In each panel A-D 200 μg of
reporter vector was co-transfected with the indicated concentration of
dsRNA. Each bar represents the average of three duplicate experiments. In
FIG. 1A, 50 nM of each dsRNA was used. In FIG. 1B, 1 nM of each dsRNA was
used. In FIG. 1C, 200 pM of each dsRNA was used. In FIG. 1D, 50 pM of
each dsRNA was used.

[0017]FIG. 5 shows superior knockout of the HNRPH1 gene by a 27-mer of the
invention as compared to a 21-mer directed to the same target. Western
blots obtained from HEK 293 cells after transfection with with
EGFP-specific siRNA (SEQ ID No. 6/7; C) (negative control) and an HNRPH1
specific 21-mer siRNA duplex (SEQ ID Nos. 51/52; 21+2) at varying
concentrations, or with an HNRPH1 specific 27-mer siRNA duplex (SEQ ID
Nos. 53/54; 27+0) at varying concentrations, as described in Example 4.

[0018]FIG. 6 shows the reaction of Dicer with various length RNA duplexes
as described in Example 5. Dicer was able to digest 25-29-mers primarily
into about a 21 basepair duplex but dd not digest the 21 nucleotide long
test duplex.

[0019]FIG. 7 shows the relative expression of EGFP after RNAi assays using
a 27-mer dsRNA versus shorter 21-mer siRNAs contained within the 27-mer
sequence as described in more detail in Example 6. As shown a blunt ended
27-mer that covers a poor site for a 21 nucleotide RNAi can effectively
target that site.

[0020]FIG. 8 shows the results of RNAi assays after treatment by various
effector dsRNA molecules and pools of molecules as set forth in Example
6.

[0021]FIG. 9 shows the time course study of the duration of the RNAi
effect with various effector molecules as described in Example 7. The
study shows the duration of the RNAi effect is at least about twice as
long with the 27-mer dsRNA of the invention as with 21-mers. The "27+0
UU" sequences are set forth in SEQ ID NOs:28 and 29. The "Mut-16"
sequences are set forth in SEQ ID NOs:70 and 71. The "Mut-16,17"
sequences are set forth in SEQ ID NOs:72 and 73. The "Mut-15,16,17"
sequences are set forth in SEQ ID NOs:74 and 75.

[0022]FIG. 10 shows the images of cells in a time course study of the
duration of the RNAi effect with various effector molecules as described
in Example 7. The study shows the duration of the RNAi effect is at least
about twice as long with the 27-mer dsRNA of the invention as with
21-mers.

[0023]FIG. 11 shows that neither interferon alpha (FIG. 11A) or interferon
beta (FIG. 11B) are induced by the 27-mer dsRNA of the invention as
described in more detail in Example 8.

[0024]FIG. 12 shows the results of a PKR activation assay in which long
dsRNA resulted in strong PKR activation (positive control) while all of
the short synthetic RNAs showed no evidence for PKR activation.

DETAILED DESCRIPTION OF THE INVENTION

[0025]The invention is directed to compositions that contain double
stranded RNA ("dsRNA"), and methods for preparing them, that are capable
of reducing the expression of target genes in eukaryotic cells. One of
the strands of the dsRNA contains a region of nucleotide sequence that
has a length that ranges from about 19 to about 23 nucleotides that can
direct the destruction of the RNA transcribed from the target gene.

[0026]For purposes of the invention a suitable dsRNA contains one
oligonucleotide sequence, a first sequence, that is at least 25
nucleotides in length and no longer than about 30 nucleotides. More
preferably this sequence of RNA is between about 26 and 29 nucleotides in
length. Still more preferably this sequence is about 27 or 28 nucleotides
in length, 27 nucleotides is most preferred. The second sequence of the
dsRNA can be any sequence that anneals to the first sequence under
biological conditions, such as within the cytoplasm of a eukaryotic cell.
Generally, the second oligonucleotide sequence will have at least 19
complementary base pairs with the first oligonucleotide sequence, more
typically the second oligonucleotides sequence will have about 21 or more
complementary base pairs, and more preferably about 25 or more
complementary base pairs with the first oligonucleotide sequence. In a
preferred embodiment the second sequence is the same length as the first
sequence.

[0027]In certain embodiments the double-stranded RNA structure the first
and second oligonucleotide sequences exist on separate oligonucleotide
strands which can be and typically are chemically synthesized. In
preferred embodiments both strands are between 26 and 30 nucleotides in
length. In one preferred embodiment both strands are 27 nucleotides in
length, are completely complementary and have blunt ends. The dsRNA can
be from a single RNA oligonucleotide that undergoes intramolecular
annealing or, more typically, the first and second sequences exist on
separate RNA oligonucleotides.

[0028]Suitable dsRNA compositions that contain two separate
oligonucleotides can be chemically linked outside their annealing region
by chemical linking groups. Many suitable chemical linking groups are
known in the art and can be used. Suitable groups will not block Dicer
activity on the dsRNA and will not interfere with the directed
destruction of the RNA transcribed from the target gene.

[0029]The first and second oligonucleotide sequences are not required to
be completely complimentary. In fact, it is preferred that the
3'-terminus of the sense strand contains one or more mismatches. It is
more preferred that two mismatches be incorporated at the 3'terminus. In
a most preferred embodiment the dsRNA of the invention is a double
stranded RNA molecule containing two RNA oligonucleotides each of which
is 27 nucleotides in length and, when annealed to each other, have blunt
ends and a two nucleotide mismatch on the 3'-terminus of the sense strand
(the 5'-terminus of the antisense strand).

[0030]One feature of the dsRNA compositions of the invention is that they
can serve as a substrate for Dicer. Typically, the dsRNA compositions of
this invention will not have been treated with Dicer, other RNAses, or
extracts that contain them. Such treatments could digest the dsRNA to
lengths of less than 25 nucleotides that are no longer Dicer substrates.
Several methods are known and can be used for determining whether a dsRNA
composition serves as a substrate for Dicer. For example, Dicer activity
can be measured in vitro using the Recombinant Dicer Enzyme Kit (GTS, San
Diego, Calif.) according to the manufacturer's instructions. Dicer
activity can be measured in vivo by treating cells with dsRNA and
maintaining them for 24 h before harvesting them and isolating their RNA.
RNa can be isolated using standard methods, such as with the RNeasy®
Kit (Qiagen) according to the manufacturer's instructions. The isolated
RNA can be separated on a 10% PAGE gel which is used to prepare a
standard RNA blot that can be probed with a suitable labeled
deoxyoligonucleotide, such as an oligonucleotide labeled with the
Starfire® Oligo Labeling System (Integrated DNA Technologies, Inc.,
Coralville, Iowa).

[0031]It has been found empirically that these longer dsRNA species of
from 25 to about 30 nucleotides give unexpectedly improved results in
terms of increased potency and increased duration of action over shorter
prior art RNAi compositions. The dsRNA compositions of the invention are
at least as active as any isolated 23 nucleotide dsRNA sequence contained
within them and in preferred embodiments more active. Without wishing to
be bound by the underlying theory of the invention, it is thought that
the longer dsRNA species serve as a substrate for the enzyme Dicer in the
cytoplasm of a cell. In addition to cleaving the dsRNA of the invention
into shorter segments, Dicer is thought to facilitate the incorporation
of a single-stranded cleavage product derived from the cleaved dsRNA into
the RISC complex that is responsible for the destruction of the
cytoplasmic RNA derived from the target gene. Studies have shown that the
cleavability of a dsRNA species by Dicer corresponds with increased
potency and duration of action of the dsRNA species.

[0032]Suitable dsRNA compositions of this invention do not induce
apoptosis in the cells in which they are used. Apoptosis or "programmed
cell death," includes any non-necrotic, cell-regulated form of cell
death, as defined by criteria well established in the art. Cells
undergoing apoptosis show characteristic morphological and biochemical
features. Once the process is triggered, or the cells are committed to
undergoing apoptosis, morphological and physiological changes include
cell shrinkage, chromatin condensation, nuclear and cytoplasmic
condensation, membrane blebbing, partitioning of cytoplasm and nucleus
into membrane bound vesicles which contain ribosomes (apoptotic bodies),
and DNA degradation into a characteristic oligonucleosomal ladder
composed of multiples of 200 base pairs, leading eventually to cell
death. In vivo, these apoptotic bodies are rapidly recognized and
phagocytized by either macrophages or adjacent epithelial cells. In
vitro, the apoptotic bodies as well as the remaining cell fragments
ultimately swell and finally lyse. This terminal phase of in vitro cell
death has been termed "secondary necrosis."

[0033]The effect that a dsRNA has on a cell can depend upon the cell
itself. In some circumstances a dsRNA could induce apoptosis or gene
silencing in one cell type and not another. Thus, it is possible that a
dsRNA could be suitable for use in one cell and not another. To be
considered "suitable" a dsRNA composition need not be suitable under all
possible circumstances in which it might be used, rather it need only be
suitable under a particular set of circumstances.

[0034]Modifications can be included in the disclosed dsRNA so long as the
dsRNA remains sufficiently chemically stable, does not induce apoptosis,
does not substantially interrupt annealing of the first and second
strands, and otherwise does not substantially interfere with the directed
destruction of the RNA transcribed from the target gene. Modifications
can be incorporated in the 3'-terminal region, the 5'-terminal region, in
both the 3'-terminal and 5'-terminal region or in some instances
throughout the sequence. With the restrictions noted above in mind any
number and combination of modifications can be incorporated into the
dsRNA. Where multiple modifications are present, they may be the same or
different. Modifications to bases, sugar moieties, the phosphate
backbone, and their combinations are contemplated.

[0035]For example, either the 3' or 5' terminal regions of the sequences
in a dsRNA can be phosphorylated or biotinylated. Examples of
modifications contemplated for the phosphate backbone include
phosphonates, including methylphosphonate, phosphorothioate, and
phosphotriester modifications such as alkylphosphotriesters, and the
like. Examples of modifications contemplated for the sugar moiety include
2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy
modifications and the like. Examples of modifications contemplated for
the base groups include abasic sugars, 2-O-alkyl modified pyrimidines,
4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil
and the like. Many other modifications are known and can be used so long
as the above criteria are satisfied

[0036]The double-stranded RNA sample can be suitably formulated and
introduced into the environment of the cell by any means that allows for
a sufficient portion of the sample to enter the cell to induce gene
silencing, if it is to occur. Many formulations for dsRNA are known in
the art and can be used so long as dsRNA gains entry to the target cells
so that it can act. For example, dsRNA can be formulated in buffer
solutions such as phosphate buffered saline solutions, liposomes,
micellar structures, and capsids. Formulations of dsRNA with cationic
lipids can be used to facilitate transfection of the dsRNA into cells.
Suitable lipids include Oligofectamine, Lipofectamine (Life
Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or
FuGene 6 (Roche) all of which can be used according to the manufacturer's
instructions.

[0037]It can be appreciated that the method of introducing dsRNA into the
environment of the cell will depend on the type of cell and the make up
of its environment. For example, when the cells are found within a
liquid, one preferable formulation is with a lipid formulation such as in
lipofectamine and the dsRNA can be added directly to the liquid
environment of the cells. Lipid formulations can also be administered to
animals such as by intravenous, intramuscular, or intraperitoneal
injection, or orally or by inhalation or other methods as are known in
the art. When the formulation is suitable for administration into animals
such as mammals and more specifically humans, the formulation is also
pharmaceutically acceptable. Pharmaceutically acceptable formulations for
administering oligonucleotides are known and can be used. In some
instances, it may be preferable to formulate dsRNA in a buffer or saline
solution and directly inject the formulated dsRNA into cells, as in
studies with oocytes. The direct injection of dsRNA duplexes

[0038]Suitable amounts of dsRNA must be introduced and these amounts can
be empirically determined using standard methods. Typically, effective
concentrations of individual dsRNA species in the environment of a cell
will be about 50 nanomolar or less 10 nanomolar or less, more preferred
are compositions in which concentrations of about 1 nanomolar or less can
be used. Even more preferred are methods that utilize a concentration of
about 200 picomolar or less and even a concentration of about 50
picomolar or less can be used in many circumstances.

[0039]The method can be carried out by addition of the dsRNA compositions
to any extracellular matrix in which cells can live provided that the
dsRNA composition is formulated so that a sufficient amount of the dsRNA
can enter the cell to exert its effect. For example, the method is
amenable for use with cells present in a liquid such as a liquid culture
or cell growth media, in tissue explants, or in whole organisms,
including animals, such as mammals and especially humans.

[0040]As is known, RNAi methods are applicable to a wide variety of genes
in a wide variety of organisms and the disclosed compositions and methods
can be utilized in each of these contexts. Examples of genes which can be
targeted by the disclosed compositions and methods include endogenous
genes which are genes that are native to the cell or to genes that are
not normally native to the cell. Without limitation these genes include
oncogenes, cytokine genes, idiotype (Id) protein genes, prion genes,
genes that expresses molecules that induce angiogenesis, genes for
adhesion molecules, cell surface receptors, proteins involved in
metastasis, proteases, apoptosis genes, cell cycle control genes, genes
that express EGF and the EGF receptor, multi-drug resistance genes, such
as the MDR1 gene.

[0041]Expression of a target gene can be determined by any suitable method
now known in the art or that is later developed. It can be appreciated
that the method used to measure the expression of a target gene will
depend upon the nature of the target gene. For example, when the target
gene encodes a protein the term "expression" can refer to a protein or
transcript derived from the gene. In such instances the expression of a
target gene can be determined by measuring the amount of mRNA
corresponding to the target gene or by measuring the amount of that
protein. Protein can be measured in protein assays such as by staining or
immunoblotting or, if the protein catalyzes a reaction that can be
measured, by measuring reaction rates. All such methods are known in the
art and can be used. Where the gene product is an RNA species expression
can be measured by determining the amount of RNA corresponding to the
gene product. Several specific methods for detecting gene expression are
described in Example 1. The measurements can be made on cells, cell
extracts, tissues, tissue extracts or any other suitable source material.

[0042]The determination of whether the expression of a target gene has
been reduced can be by any suitable method that can reliably detect
changes in gene expression. Typically, the determination is made by
introducing into the environment of a cell undigested dsRNA such that at
least a portion of that dsRNA enters the cytoplasm and then measuring the
expression of the target gene. The same measurement is made on identical
untreated cells and the results obtained from each measurement are
compared. When the method appears to reduce the expression of the target
gene by about 10% or more (which is equivalent to about 90% or less) of
the level in an untreated organism, for purposes of this invention, the
method is considered to reduce the expression of the target gene.
Typically, the method can be used to reduce the expression of a target
gene by far more than 10%. In some instances the method can be used to
reduce the expression by about 50% or more, in more preferred methods the
expression is reduced by about 75% or more, still more preferable are
methods that reduce the expression by about 90% or more, or even about
95% or more, or about 99% or more or even by completely eliminating
expression of the target gene.

[0043]The dsRNA can be formulated as a pharmaceutical composition which
comprises a pharmacologically effective amount of a dsRNA and
pharmaceutically acceptable carrier. A pharmacologically or
therapeutically effective amount refers to that amount of a dsRNA
effective to produce the intended pharmacological, therapeutic or
preventive result. The phrases "pharmacologically effective amount" and
"therapeutically effective amount" or simply "effective amount" refer to
that amount of an RNA effective to produce the intended pharmacological,
therapeutic or preventive result. For example, if a given clinical
treatment is considered effective when there is at least a 20% reduction
in a measurable parameter associated with a disease or disorder, a
therapeutically effective amount of a drug for the treatment of that
disease or disorder is the amount necessary to effect at least a 20%
reduction in that parameter.

[0044]The phrase pharmaceutically acceptable carrier refers to a carrier
for the administration of a therapeutic agent. Exemplary carriers include
saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. For drugs administered orally, pharmaceutically
acceptable carriers include, but are not limited to pharmaceutically
acceptable excipients such as inert diluents, disintegrating agents,
binding agents, lubricating agents, sweetening agents, flavoring agents,
coloring agents and preservatives. Suitable inert diluents include sodium
and calcium carbonate, sodium and calcium phosphate, and lactose, while
corn starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent, if
present, will generally be magnesium stearate, stearic acid or talc. If
desired, the tablets may be coated with a material such as glyceryl
monostearate or glyceryl distearate, to delay absorption in the
gastrointestinal tract. The pharmaceutically acceptable carrier of the
disclosed dsRNA composition may be micellar structures, such as a
liposomes, capsids, capsoids, polymeric nanocapsules, or polymeric
microcapsules.

[0045]Polymeric nanocapsules or microcapsules facilitate transport and
release of the encapsulated or bound dsRNA into the cell. They include
polymeric and monomeric materials, especially including
polybutylcyanoacrylate. A summary of materials and fabrication methods
has been published (see J. Kreuter, (1991) Nanoparticles-preparation and
applications. In: M. Donbrow (Ed.) Microcapsules and nanoparticles in
medicine and pharmacy. CRC Press, Boca Raton, Fla., pp. 125-14). The
polymeric materials which are formed from monomeric and/or oligomeric
precursors in the polymerization/nanoparticle generation step, are per se
known from the prior art, as are the molecular weights and molecular
weight distribution of the polymeric material which a person skilled in
the field of manufacturing nanoparticles may suitably select in
accordance with the usual skill.

[0046]Suitably formulated pharmaceutical compositions of this invention
can be administered by any means known in the art such as by parenteral
routes, including intravenous, intramuscular, intraperitoneal,
subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical
(including buccal and sublingual) administration.

[0047]In general a suitable dosage unit of dsRNA will be in the range of
0.001 to 0.25 milligrams per kilogram body weight of the recipient per
day, preferably in the range of 0.01 to 20 micrograms per kilogram body
weight per day, more preferably in the range of 0.01 to 10 micrograms per
kilogram body weight per day, even more preferably in the range of 0.10
to 5 micrograms per kilogram body weight per day, and most preferably in
the range of 0.1 to 2.5 micrograms per kilogram body weight per day.
Preferably, pharmaceutical composition comprising the dsRNA is
administered once daily. However, the therapeutic agent may also be dosed
in dosage units containing two, three, four, five, six or more sub-doses
administered at appropriate intervals throughout the day. In that case,
the dsRNA contained in each sub-dose must be correspondingly smaller in
order to achieve the total daily dosage unit. The dosage unit can also be
compounded for a single dose over several days, e.g., using a
conventional sustained release formulation which provides sustained and
consistent release of the dsRNA over a several day period. Sustained
release formulations are well known in the art. In this embodiment, the
dosage unit contains a corresponding multiple of the daily dose.
Regardless of the formulation, the pharmaceutical composition must
contain dsRNA in a quantity sufficient to inhibit expression of the
target gene in the animal or human being treated. The composition can be
compounded in such a way that the sum of the multiple units of dsRNA
together contain a sufficient dose.

[0048]Data can be obtained from cell culture assays and animal studies to
formulate a suitable dosage range for humans. The dosage of compositions
of the invention lies, preferably, within a range of circulating
concentrations that include the ED50 (as determined by known methods)
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of administration
utilized. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to achieve a
circulating plasma concentration range of the compound that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses in
humans. Levels of dsRNA in plasma may be measured by standard methods,
for example, by high performance liquid chromatography.

[0049]The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its scope.

[0052]The purity of each oligomer was determined by capillary
electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.,
Fullerton, Calif.). The CE capillaries had a 100 μm inner diameter and
contained ssDNA 100R Gel (Beckman-Coulter). Typically, about 0.6 nmole of
oligonucleotide was injected into a capillary, ran in an electric field
of 444 V/cm and detected by UV absorbance at 260 nm. Denaturing
Tris-Borate-7 M-urea running buffer was purchased from Beckman-Coulter.
Oligoribonucleotides were at least 90% pure as assessed by CE for use in
experiments described below. Compound identity was verified by
matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)
mass spectroscopy on a Voyager DE® Biospectometry Work Station
(Applied Biosystems, Foster City, Calif.) following the manufacturer's
recommended protocol. Relative molecular masses of all oligomers were
within 0.2% of expected molecular mass.

[0054]Nomenclature. For consistency, the following nomenclature has been
employed throughout the Examples. Names given to duplexes indicate the
length of the oligomers and the presence or absence of overhangs. A
"21+2" duplex contains two RNA strands both of which are 21 nucleotides
in length, also termed a 21-mer siRNA duplex, and having a 2 base
3'-overhang. A "21-2" design is a 21-mer siRNA duplex with a 2 base
5'-overhang. A 21-0 design is a 21-mer siRNA duplex with no overhangs
(blunt). A "21+2UU" is a 21-mer duplex with 2-base 3'-overhang and the
terminal 2 bases at the 3'-ends are both U residues (which may result in
mismatch with target sequence).

Example 2

[0055]This example demonstrates that dsRNAs having strands that are 25
nucleotides in length or longer have surprisingly increased potency in
mammalian systems than known 21-23-mer siRNAs.

[0056]Cell Culture, Transfection, and EGFP Assays. Human embryonic kidney
(HEK) 293 cells were grown in DMEM medium supplemented with 10% fetal
bovine serum (FBS) (Irvine Scientific, Santa Ana, Calif.). Transfections
were done at 90% confluence in 24-well plates using Lipofectamine 2000
(Invitrogen, Carlsbad, Calif.) according to the manufacturer's
instructions. Briefly, 50 μl of Opti-MEM media was mixed with nucleic
acids, including siRNA duplexes and/or 100-200 ng plasmid pEGFP-C1
(Clontech, Palo Alto, Calif.) for 5 min. Nucleic acids were then mixed
with 50 μl of Opti-MEM media that had been pre-mixed with 1.5 μl of
Lipofectamine 2000 and incubated at room temperature for 15 min. The
lipid-nucleic acid mixtures were added to cells after removal of old
media and swirled and then an additional 0.4 ml of media pre-warmed to
37° C. was added. Incubation was continued at 37° C. and
cells were assayed for fluorescence at the times indicated. Each assay
was performed in triplicate. EGFP expression levels were measured by
direct fluorescence in a fluorescence-activated cell sorter (FACS)
(Moslo-MLS, Dako Cytomation, Fort Collins, Colo.) in the City of Hope
Cytometrics Core Facility (Duarte, Calif.). EGFP expression was measured
as the percentage of cells showing detectable fluorescence above
background (mock-transfected negative control cells).

[0057]NIH 3T3 cells that stably expressed EGFP (Kim and Rossi, 2003,
Antisense Nucleic Acid Drug Dev., 13:151-155) were grown in DMEM media
supplemented with 10% FBS. Cells were plated at 30% density on 24-well
plates and transfected with siRNA alone without reporter plasmid using
the same method described above. Media was changed at 24 h and EGFP
assays were performed at 3, 6 and 9 days post-transfection. At 3 days
post-transfection, 1×105 cells were used for extract
preparation and 1×104 cells were re-plated and continued
incubation for later assay. At day 6, 1×105 cells were used
for extract preparation, and 1×104 cells were re-plated and
continued incubation for later assay. At day 9, 1×105 cells
were used for extract preparation. For extract preparation,
1×105 cells were suspended in 300 μl phosphate buffered
saline (PBS) and sonicated for 10 sec. Cells were centrifuged at 14,000 g
for 2 min and cell supernatant was recovered for fluorometry. EGFP
fluorescence was examined using a VersaFluor Cuvette Fluorometer
(Bio-Rad, Hercules, Calif.) using excitation filter D490 and emission
filter D520. Percentage of EGFP expression was determined relative to
extract prepared from non-transfected control cells.

[0058]In addition, cells were directly examined by fluorescence microscopy
using a Nikon Eclipse TE2000-S (Nikon Instech Co., Kanagawa, JP) using
the program Spot v3.5.8. Images were digitally captured with identical
exposure times so that comparisons between cells samples could be made.

[0059]Nucleic Acid Reagents. The reporter system employed EGFP either as a
transfection plasmid vector pEGFP-C1 (Clontech, Palo Alto, Calif.) or as
a stable transformant in an NIH 3T3 cell line. The coding sequence of
EGFP is shown below, from Genbank accession #U55763. The ATG start codon
and TAA stop codons are highlighted in bold font and sites target by
siRNA reagents are underscored.

[0062]RNA duplexes were synthesized and prepared as described in Example
1. RNA duplexes targeting EGFP Site-1 are summarized in Table 1 below.
Some sequences had the dinucleotide sequence "UU" placed at the 3'-end of
the sense strand (Elbashir et al., 2001, EMBO J., 20:6877-6888; Hohjoh,
2002, FEBS Lett., 521:195-199). Mismatches that resulted from including
3'-terminal "UU" or where a mismatch was intentionally positioned are
highlighted in bold and underscored.

[0065]This example demonstrates that the longer dsRNAs of the invention
have about a 100-fold or more higher potency than traditional 21-mer
siRNAs. The enhanced effect was first seen at about a 25-mer length and
maximal potency was achieved with a 27-mer. Potent RNAi effects were
observed for 30-mer duplexes (the longest compounds tested herein), with
no apparent toxicity to either the HEK 293 cells or NIH 3T3 cells.
Furthermore, as duplex length was increased above 25-mer length
(presumably when the duplex is sufficiently long to be a Dicer
substrate), a 2-base 3'-overhang (as taught in the prior art) is no
longer necessary. In the present experiments 25-mer duplexes with a
2-base 5'-overhang had similar potency as did blunt ended duplexes or
duplexes with a 2-base 3'-overhang. In the current experimental system,
the 27-mer blunt duplex showed greatest potency.

[0066]Within the set of 27-mer RNA duplexes tested in this example,
duplexes that included base mismatches between the sense and antisense
strands (SEQ ID Nos. 30/31, 32/33, 34/35) were more potent than the
duplex having perfect complementarity (SEQ ID Nos. 28/29). These duplexes
(27+0UU, 27+2UU, and 27-2UU) had 1 or 2 mismatches at the 3'-end of the
sense strand. The set of 27-mer duplexes were compared for effective
suppression of EGFP expression in the HEK 293 cell transient transfection
assay and the results are shown in FIG. 3. Duplex 27+0UU (SEQ ID Nos.
30/31) was most potent.

[0067]The use of mismatches or decreased thermodynamic stability
(specifically at the 3'-sense/5'-antisense position) has been proposed to
facilitate or favor entry of the antisense strand into RISC (Schwarz et
al., 2003, Cell, 115:199-208; Khvorova et al., 2003, Cell, 115:209-216),
presumably by affecting some rate-limiting unwinding steps that occur
with entry of the siRNA into RISC. Because of this terminal base
composition has been included in design algorithms for selecting active
21-mer siRNA duplexes (Ui-Tei et al., 2004, Nucleic Acids Res.,
32:936-948; Reynolds et al., 2004, Nat. Biotechnol., 22:326-330). It has
been proposed that the 27-mer duplexes employed in this example do not
directly enter RISC but first are cleaved by Dicer into 21-mer siRNAs.
With Dicer cleavage, the small end-terminal sequence which contains the
mismatches will either be left unpaired with the antisense strand (become
part of a 3'-overhang) or be cleaved entirely off the final 21-mer siRNA.
These "mismatches", therefore, do not persist as mismatches in the final
RNA component of RISC. It was surprising to find that base mismatches or
destabilization of segments at the 3'-end of the sense strand of a Dicer
substrate improve the potency of synthetic duplexes in RNAi, presumably
by facilitating processing by Dicer.

Example 3

[0068]This example demonstrates that the use of 25-30 nucleotide RNA
duplexes allows gene targeting at a site that could not be effectively
targeted using traditional siRNA 21-mer designs.

[0069]It is currently expected in the art that the majority of 21-mer
siRNA duplexes targeted to sites within a given target gene sequence will
be ineffective (Holen et al., 2002, Nucleic Acids Res., 30:1757-1766).
Consequently, a variety of sites are commonly tested in parallel or pools
containing several distinct siRNA duplexes specific to the same target
with the hope that one of the reagents will be effective (Ji et al.,
2003, FEBS Lett., 552:247-252). To overcome the need to pool or engage in
large scale empiric testing, complex design rules and algorithms have
been devised to increase the likelihood of obtaining active RNAi effector
molecules (Schwarz et al., 2003, Cell, 115:199-208; Khvorova et al.,
2003, Cell, 115:209-216; Ui-Tei et al., 2004, Nucleic Acids Res.,
32:936-948; Reynolds et al., 2004, Nat. Biotechnol., 22:326-330). These
design rules significantly limit the number of sites amenable to RNAi
knockdown within a given target gene. In fact, the design can be overly
restrictive in situations demanding the suppression of specific alleles
or isoforms. Moreover, the rules are not perfect and do not always
provide active siRNA effector molecules. This example shows that the use
of dsRNA duplexes of the present invention allow RNAi targeting at sites
that were ineffectively targeted by previously known 21-mer siRNA
reagents. This result minimizes the need for empirically testing multiple
sites or using pooled reagent sets.

[0070]Nucleic Acid Reagents. The reporter system employed EGFP as in SEQ
ID No. 1 above. Site-2 in EGFP, as shown in Example 1, was targeted. RNA
duplexes were synthesized and prepared as described in Example 1. RNA
duplexes targeting EGFP Site-2 are summarized in Table 2 below. Duplex
EGFPS2-27+0 mm was a blunt 27-mer duplex with a 2 base mismatch at the
terminal 2 bases of the sense strand. These bases are shown in bold and
underscored.

[0071]Results. HEK 293 cells were mock transfected (negative control),
transfected with 200 ng EGFP reporter plasmid alone (positive control),
or reporter plasmid+RNA duplexes at varying concentrations as described
previously. EGFP expression was assessed using the FACS assay at 24 h
post-transfection. Results are shown in FIG. 4. At 10 nM concentration,
the traditional 21-mer siRNA duplex with 2-base 3'-overhangs targeted to
Site-2 in the EGFP gene (SEQ ID Nos. 44/45) did not detectably reduce
EGFP expression. In contrast, a 10 nM concentration of the longer 27-mer
duplex RNA (SEQ ID No. 46/47) reduced EGFP by about 80% or more and 10 nM
of a related 27-mer (SEQ ID No. 48/49) reduced EGFP by about 90% or more.
As in Example 2 above, 3'- or 5'-overhangs did not improve activity over
the blunt ended version. The 27-mer with the 2-base mismatch in the sense
strand (SEQ ID Nos. 48/49) showed improved activity as compared to the
perfectly matched 27-mer (SEQ ID Nos. 46/47). It is possible that
destabilization of the RNA duplex at this position improves efficiency of
cleavage by Dicer.

[0072]This example demonstrates that dsRNAs of the invention can
efficiently target sites within the EGFP gene that were previously
considered poor targets by previously known methods. Use of the method of
the invention will therefore simplify site selection and design criteria
for RNAi. This example also shows that the intentional placement of
mismatches at the 3'-terminus of the sense strand increases the potency
of the 27-mer duplex.

Example 4

[0073]This example demonstrates the use of the disclosed dsRNA duplexes to
reduce expression of the human HNRPH1 gene in HEK 293 cells.

[0074]Western Blot. HEK 293 cells were cultured in a 6-well plate. At 30%
confluence, cells were transfected with iRNA duplexes as outlined in
Example 2 above except that all reagents were used at 5-fold higher
volume due to the larger scale of the cultures. Cells were harvested at
72 h in 300 μl phosphate buffered saline (PBS) and sonicated for 10
sec. Cell lysates was centrifuged for 2 min at 14,000 g and the
supernatant was collected. Aliquots of 2 μl were taken from the
cleared lysates which were run on a 10% SDS-PAGE gel. The HNRPH1 gene
product was detected using a rabbit polyclonal anti-HNRPH1 antiserum and
an anti-rabbit antibody conjugated with alkaline phosphatase (Sigma, St.
Louis, Mo.). As control, β-actin was detected by a murine anti-human
actin antibody (Sigma, St. Louis, Mo.) and anti-mouse antibody conjugated
with alkaline phosphatase (Sigma, St. Louis, Mo.), as previously
described (Markovtsov et al., 2000, Mol. Cell Biol., 20:7463-79).

[0077]HEK 293 cells were transfected with EGFP-specific siRNA (SEQ ID No.
6/7) (negative control) and an HNRPH1 specific 21-mer siRNA duplex (SEQ
ID Nos. 51/52) at varying concentrations, or with an HNRPH1 specific
27-mer siRNA duplex (SEQ ID Nos. 53/54) at varying concentrations, as
described previously. HNRPH1 expression was assessed by Western Blot
assay at 72 h post-transfection. Results are shown in FIG. 5. As shown,
only a slight decrease in HNRPH1 protein levels occurred after treatment
with 20 nM of the 21-mer siRNA (SEQ ID Nos 51/52) while significant
inhibition was seen using 1 nM of the 27-mer dsRNA (SEQ ID Nos 53/54) and
almost complete elimination of HNRPH1 protein was achieved using 5 nM of
the 27-mer RNA duplex. Improved reduction in gene expression by RNAi
methods is therefore also seen for human genes using the method of the
invention.

Example 5

[0078]This example demonstrates a method for determining whether a dsRNA
serves as s substrate for Dicer.

[0079]In vitro Dicer assay. Recombinant human Dicer enzyme (Gene Therapy
Systems, San Diego, Calif.) was incubated with synthetic duplex RNA
oligonucleotides according to the manufacturer's instructions. Briefly, 2
units of Dicer was incubated in a buffer supplied by the manufacturer
with 250 pmoles of RNA duplex in a 50 μl volume (5 μM RNA
concentration) for 18 h at 37° C. Half of each reaction was
separated on non-denaturing PAGE (10% acrylamide) and visualized using
ethidium bromide staining with UV excitation.

[0081]This example shows that the longer RNA duplexes used in the method
of the invention are substrates for the Dicer endoribonuclease.

Example 6

[0082]This example demonstrates that 27-mer duplexes have more RNAi
activity than any of the shorter 21-mer duplexes that they encompass.

[0083]Theoretically, a variety of short 21-mer siRNAs could result from
the action of Dicer on longer duplex RNAs. For example, based upon the
antisense strand, 7 different 21-mer species could result from
degradation of a 27-mer sequence. It is possible that one of these
21-mers (or a combination of 21-mers) accounts for the activity observed
with the previously tested 27-mer dsRNA. This example shows that no
single 21-mer duplex or mixture of 21-mers resulting from degradation of
a 27-mer sequence functions as effectively as its parent 27-mer duplex at
reducing EGFP expression.

[0084]Nucleic Acid Reagents. RNA duplexes were prepared as described in
Example 1. The sequences of a set of 21-mer RNA duplexes from within EGFP
Site-1 were prepared. The duplexes are listed below in Table 4. The
21-mer duplexes are aligned beneath the parent 27-mer to illustrate their
relative positioning. The 27-mer blunt duplex (SEQ ID No. 28/29) and the
21-mer duplex 21+2(7) (SEQ ID No. 6/7) are shown in Example 2 (Table 1)
and were also used.

[0085]The each of the 21-mer duplexes from Table 4 was transfected
individually or together as a pool into HEK 293 cells with 200 ng of EGFP
reporter plasmid as described previously. The result from each
transfection was compared with the 27-mer duplex (SEQ ID No. 28/29). The
relative EGFP expression from each experiment is shown in FIG. 7. At
concentrations of 50 or 200 pM, none of the individual 21-mer duplexes or
the pooled set of 7 21-mer duplexes showed activity comparable with the
27-mer duplex. For pools, 50 pM and 200 pM represent the total
concentration of all RNAs transfected together, rather than for
individual duplexes. FIG. 7 shows that the potency of the 27-mer duplex
was much higher than for any of the shorter 21-mer sequences, which
included every possible 21-mer duplex that could result from degradation
of the parent 27-mer.

[0086]The activity of "diced" products made from digestion of the 27-mer
duplex with recombinant Dicer enzyme in vitro was compared with the
parent 27-mer compound in RNAi assays. The 27-mer duplex (SEQ ID No.
28/29) was degraded using Dicer as described in Example 5 above and
fragments ("diced" products) were diluted and directly used in
transfection experiments. EGFP expression levels were measured following
transfection of HEK 293 cells with 200 ng EGFP reporter plasmid with a
21-mer duplex (SEQ ID No. 6/7), a 27-mer duplex (SEQ ID No. 28/29),
products of in vitro Dicer degradation ("diced" products), a mutant
27-mer with 4 base central mismatch (SEQ ID No. 36/37), and the pooled
set of 7 21-mer duplexes (SEQ ID Nos. 6/7 and 55/56, 57/58, 59/60, 61/62,
63/64, 65/66). Results are shown in FIG. 8. Again, the 27-mer duplex was
the most potent reagent in reducing EGFP expression. The "diced" products
were more effective than the set of pooled 21-mer duplexes. One
explanation for this result is that the in vitro dicing reaction was
incomplete and some intact 27-mer remains even after 18 h incubation
(residual 27-mer is seen in FIG. 6).

[0087]This example provides another demonstration that the improved
potency of dsRNA 27-mers is not derived from a highly active individual
or a pooled set of short 21-mer duplexes.

Example 7

[0088]This example demonstrates that gene suppression using the 27-mer
duplexes of the invention last twice as long as suppression achieved
using 21-mer duplexes.

[0089]Suppression of gene expression using synthetic siRNA typically has a
duration of 3-4 days in tissue culture (Chiu and Rana, 2002, Mol. Cell,
10:549-561). Methods that increase the duration of the RNAi effect would
improve the functional utility of RNAi as an experimental tool in tissue
culture and would be beneficial for use of RNAi in vivo.

[0090]NIH 3T3 cells were transfected with 5 nM of either the 21-mer duplex
(SEQ ID No. 6/7) or the 27-mer duplex (SEQ ID No. 30/31). Cell extracts
were prepared and measured for EGFP protein expression in a cuvette
fluorometer (as described in Example 2 above) at 2, 4, 6, 8, and 10 days
post-transfection. Results are shown in FIG. 9. In addition, images of
cells that were transfected in parallel using 1 nM siRNAs were obtained
using fluorescence microscopy (as described in Example 2) and the images
are shown in FIG. 10. EGFP expression was suppressed to about 70% of
control levels at day 4 but returned to about 80% of control levels at
day 6 and was at control levels at day 8. In contrast, suppression using
the 27-mer duplex was about 90% or more at day 8 and was still at about
70% on day 10.

[0091]The 27-mer duplexes used in the present example demonstrates
suppression of gene expression for at least twice the duration seen using
21-mer duplexes.

Example 8

[0092]This example demonstrates that the dsRNA duplexes of the invention
do not activate the interferon response.

[0093]Historically, long double stranded RNA was considered to be
ineffective as an agent for reducing gene expression in mammalian cells
because it tends to activate interferon pathway responses and lead to a
variety of metabolic disturbances in cells which are not sequence
specific. Short 21-mer siRNAs were considered useful for RNA I
experiments because, in addition to suppressing gene expression, they
avoid interferon activation. Because the more active double stranded RNAs
of this invention are longer than known siRNA duplexes, they were
examined further to show that they do not activate interferon. Duplexes
of up to about 30-mer lengths were tested.

[0096]Results. HEK 293 cells were transfected with ssRNA, 21-mer duplex,
27-mer duplex, or no RNA (negative control, mock transfection) as
described above. As shown in FIG. 11, high levels of interferon alpha
(FIG. 11A) and interferon beta (FIG. 11B) were detected after
transfection with ssRNA however no interferon was detected when 21-mer or
27-mer RNAs were transfected.

[0098]We conclude that the longer synthetic RNAs used in the invention for
improved RNAi mediated suppression of gene expression do not activate
interferon responses and therefore should be usable in a wide variety of
mammalian systems.

Example 9

[0099]This example demonstrates a method for determining an effective dose
of the dsRNA of the invention in a mammal. A therapeutically effective
amount of a composition containing a sequence that encodes a dsRNA,
(i.e., an effective dosage), is an amount that inhibits expression of the
product of the target gene by at least 10 percent. Higher percentages of
inhibition, e.g., 20, 50, 90 95, 99 percent or higher may be preferred in
certain circumstances. Exemplary doses include milligram or microgram
amounts of the molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 5 milligrams per kilogram, about
100 micrograms per kilogram to about 0.5 milligrams per kilogram, or
about 1 microgram per kilogram to about 50 micrograms per kilogram). The
compositions can be administered one or more times per week for between
about 1 to 10 weeks, e.g., between 2 to 8 weeks, or between about 3 to 7
weeks, or for about 4, 5, or 6 weeks, as deemed necessary by the
attending physician. Treatment of a subject with a therapeutically
effective amount of a composition can include a single treatment or a
series of treatments.

[0100]Appropriate doses of a particular dsRNA composition depend upon the
potency of the molecule with respect to the expression or activity to be
modulated. One or more of these molecules can be administered to an
animal, particularly a mammal, and especially humans, to modulate
expression or activity of one or more target genes. A physician may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular subject will depend upon a variety of other factors including
the severity of the disease, previous treatment regimen, other diseases
present, off-target effects of the active agent, age, body weight,
general health, gender, and diet of the patient, the time of
administration, the route of administration, the rate of excretion, any
drug combination, and the degree of expression or activity to be
modulated.

[0101]The efficacy of treatment can be monitored by measuring the amount
of the target gene mRNA (e.g. using real time PCR) or the amount of
product encoded by the target gene such as by Western blot analysis. In
addition, the attending physician can monitor the symptoms associated
with the disease or disorder afflicting the patient and compare with
those symptoms recorded prior to the initiation of treatment

[0102]All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the same
extent as if each reference were individually and specifically indicated
to be incorporated by reference and were set forth in its entirety
herein.

[0103]The use of the terms "a" and "an" and "the" and similar referents in
the context of describing the invention (especially in the context of the
following claims) are to be construed to cover both the singular and the
plural, unless otherwise indicated herein or clearly contradicted by
context. The terms "comprising," "having," "including," and "containing"
are to be construed as open-ended terms (i.e., meaning "including, but
not limited to,") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range, unless
otherwise indicated herein, and each separate value is incorporated into
the specification as if it were individually recited herein. All methods
described herein can be performed in any suitable order unless otherwise
indicated herein or otherwise clearly contradicted by context. The use of
any and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the invention and does
not pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of the
invention.

[0104]Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become apparent
to those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the invention to
be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described elements
in all possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by context.